T cell responses are controlled by intracellular signals emanating from ligation of cell surface receptors. While the concept of a second signal has been established for many years, with CD28 representing the initial provider of this second signal, it is clear that signals from several other co-stimulatory receptors are needed for an effective and persistent T cell response. We have extensively studied a member of the TNFR family, namely OX40 (CD134), whose expression is induced on T cells several hours to days after the initial antigen recognition event. We have demonstrated that 0X40 costimulation prolongs clonal expansion of CD4 and CDS T cells by regulating cell cycle progression;enhances T cell differentiation and cytokine secretion particularly toward the Th2 phenotype;and furthermore promotes T cell survival by antagonizing apoptosis. Multiple in vivo studies in varying models of disease over the past 5 or more years have reinforced these conclusions and led to the concept that the synergistic and temporal activities of CD28 and OX40 represent key events in most T cell mediated immune responses. In this renewal application, we seek to extend our functional data and further define molecularly the mode of action of OX40 and how OX40 signals integrate with antigen and CD28 signals. We have shown several common intracellular targets of OX40 including novel molecules survivin and aurora B kinase that regulate G1-S cell cycle progression, and Bcl-2, Bcl-xL, and Bfl-1 that control survival during and after the phase of T cell division. We have developed highly physiological and novel transgenic and retroviral systems to understand regulation of OX40 in primary T cells. We will investigate how OX40 leads to activation of Akt and NF-KB and the extent that these signaling moieties co-operate in promoting cell division and survival of T cells. Specifically, we will determine whether OX40 utilizes PKC0, PDK1, and MEK1/ERK to target Akt and NF-KB, and how these signaling cascades impact the expression and function of survivin and aurora B kinase in regulating division, and the Bcl-2 anti-apoptotic molecules in regulating survival. We will further define how OX40 targets NFATd nuclear accumulation leading to IL-4 secretion and Th2 differentiation, and then define signaling complexes induced by OX40 at the T cell membrane in lipid rafts. Lastly, we will use several models of in vivo T cell response involving lung inflammation and anti-tumor activity to extend the in vitro signaling analyses and understand molecularly how co-stimulatory signaling impacts physiological T cell function. These studies are tremendously important in defining the action of OX40 and the integration of co-stimulatory signals, and in understanding which molecular pathways are necessary and sufficient for promoting a long-lived T cell response.